In the last decade the role of oxygen free radicals as well as other oxygen reactive metabolites has been at least somewhat elucidated. These reactive metabolites are today considered to be responsible for oxidative toxicity in mammals and resulting the biological damage Halliwell, B. and Gutteridge, J. M. C. (1989) Free Rad. Biol. Med. (2nd ed.) Clarendon Press, Oxford; Halliwell, B. (1989) Br. J. Exp. Path. 70: 737-752!.
Free radicals are defined as an atom, group of atoms or molecule which contain at least one unpaired electron in their outer shell. The oxygen molecule itself, containing two unpaired electrons is, by definition, a free radical (.sup.3 .SIGMA..sub.g O.sub.2 --biradical), although it is not a reactive one due to the arrangement of its spins it its outer shell. Reduction of the oxygen molecule is a process which does not require changes in the spin direction of movement and therefore can be easily performed.
The complete reduction of the oxygen molecule results, at the first stage, in the production of the ion radical superoxide (O.sub.2.sup.-), followed by the production of hydrogen perxide (H.sub.2 O.sub.2) which is not a free radical, although a reactive species. Addition of another electron to the hydrogen peroxide leads to the production of the hydroxyl radical (OH*), a highly reactive species Halliwell and Gutteridge (1989) ibid.!. Such a process occurs in the mitochondria for energy generation Halliwell (1989) ibid.!. In this system which contains cytochrome oxidase, the oxygen is reduced in stages. During the reduction of the oxygen molecule, active metabolites are produced. These active oxygen species can leak to the immediate surroundings and may cause biological damage.
The production of oxygen reactive metabolites is not unique to the mitochondria organ, but occurs also in many other systems. Phagocytes, for example, are known for their ability to produce the superoxide radical and other species which participate in the defence mechanism against invaders (phgocytosis) Halliwell and Gutteridge (1989) ibid.!. This process, although necessary, can also lead to biological damage in the surrounding environment. Production of the superoxide radical has been identified in liver Kupffer cells, in monocytes, basophyls, eosinofils and mast cells. In some genetic disorders, such as chronic granulomatosus disease (CGD), the phagocytes are incapable of producing the oxygen radicals and consequently the patients suffer from recurring infections which can lead to death Johnston, Jr., R. B. et al. (1975) J. Clin. Invest. 55: 1357!. Another source for free radicals are enzymes. During their catalytic activity many enzymes such as prolyl hydroxylase, lipoxygenase and cyclooxygenase, produce oxygen free radicals Halliwell and Gutteridge (1989) ibid.!.
Amongst the diseases in which free radicals have been shown to play an important role in the initiation and pathogenesis, one can find chronic inflammation, autoimmune diseases such as Hashimoto's thyroiditis, systemic lupus erythematosus, myasthenia gravis, chronic autoimmune gastritis, dermatomyositis, etc. Gutteridge, J. M. C. (1993) Free Rad. Res. Comms. 19: 141-158!. Release of a free radicals efflux by the phagocytes can increase the severity of these diseases. This is the case, for example, in rheumatoid arthritis which is characterized by chronic inflammation of the joints. In this disease, the production of the inter-joint fluid is normal, but its viscosity is changed. The major constitutent of the inter-joint fluid, hyaluronic acid, is broken down into short fragments by the oxygen reactive species Halliwell and Gutteridge (1989) ibid.!.
It has been proposed that free radicals are involved in the damage caused to the brain in brain degenerative diseases such as epilepsy, Parkinson's disease, Wilson's disease (the excess of copper in this disorder causes the transformation of the superoxide radical into a more reactive species, the hydroxyl radical, in the metal-mediated Haber-Weiss reaction) Halliwell, B. and Gutteridge J. M. C. (1984) Biochem. J. 219: 1-14!. It has also been suggest that free radicals are involved in eye diseases such as cataract and retinopathy Taylor, A. and Davies, K. J. A. (1987) Free Rad. Biol. Med. 3: 371-377!.
It has recently been demonstrated that oxygen driven free radicals play an important role in the post-ischemic damage to various biological tissues such as heart and brain. It has further been suggested that these metabolites take part in the aging process and age-related diseases such as amyloid generation, age pigmentation, neuron damage Gutteridge (1993) ibid.!
Exposure of humans to free radicals is not limited to the endogenous oxygen free radical, but also to exogenous sources. Various agrochemicals can serve as free radical generation systems as in the case of the herbicide Paraquat Kohen, R. and Chevion, M. (1985) Biochem. Pharmacol. 34: 1841-1843!. Other substances such as alloxan, isouramil, cigarette smoke, air pollutants, carcinogenic and mutagenic agents and many drugs can generate oxygen free metabolites and cause biological damage.
Exposure of living cells to continuous efflux of oxygen free radicals and reactive species led to their adaptation to living in an aerobic atmosphere. This adaptation process led to the development of several defence lines against the damage induced by these metabolites Halliwell and Gutteridge (1989) ibid.; Ames, B. N. (1983) Science 221: 1256-1264!. The defence systems vary from one species to another and between different tissues of same species.
The broad definition of an antioxidant includes compounds which can cope with the oxidative stress by various mechanisms. Amongst the different kinds, one can find compounds which can protect fatty acids against oxidation and compounds which protect proteins, DNA and other important macromolecules. The different antioxidants can be classified as follows: compounds which donate hydrogen to the damaged target; compounds which scavenge free radicals; compounds which can bind the oxidants and remove them from the target; compounds which can convert reactive species to non-reactive metabolites; compounds which can protect through stabilization of biological membranes; reducing compounds which react with the oxidants; and compounds which are capable of binding mediators, such as transition metal ions, and prevent them from participating in the Haber-Weiss reaction Kohen, R., et al. (1988) Proc. Natl. Acad. Sci. USA 85: 3175-3179!.
In contrast to the antioxidant enzymes and some other antioxidants which are produced in humans, the antioxidant vitamins (E, C and A) are present only in the diet. New molecules which possess antioxidant activity were recently found in the muscle and brain Kohen et al. (1988) ibid.!. Vitamins E and C, although not synthesized by humans, are essential for the functioning of many systems Halliwell and Gutteridge (1989) ibid.!. Elimination of these antioxidants from the diet results in severe pathological symptoms. It has been shown in many pathological cases that administration of these antioxidants results in significant improvement Halliwell and Gutteridge (1984) ibid.!. Patients undergoing irradiation therapy are given vitamin C in order to reduce some of the side effects involved with radiation. Administration of vitamin E and 2-mercaptoethylamine to rats resulted in a 30% increase of their life span Herman, D. (1982) The Free Radical Theory of Aging. In: Free Radicals in Biology, Vol. V (ed. W. A. Pryor) p. 255, Academic Press, London; Herman, D. J. (1968) Gerontology 23: 476-450!.
The enormous problems involved in using the se compounds, the difficulties in delivering them to their targets and the lack of knowledge in developing pharmaceutical dosage forms for these antioxidants, prevent their wide use in the treatment of diseases.
In order to prevent oxidative damage and due to the high reactivity of the oxygen reactive species, antioxidant molecules have to be present at the site of their biologocal targets in high concentrations and for long periods of time. To fulfill these requirements, sustained release dosage forms of antioxidant compounds are desirable.
Since the creation of the first pharmaceutical dosage forms in the early sixties, there has been a tremendous increase in the theoretical and practical development of such forms. The progress achieved in the treatment of many cancer forms over the last decade led to the urgent need for delivery and direction of drugs to their targets. There is considerable evidence that a constant level of the drug in its biological target results in decrease in the immediate and long term side effects and improves the therapeutical effects.
In contrast to conventional dosage forms, where the drug is released immediately after its administration, the advanced dosage forms allow the drug to be released in unique manners, as follows: (1) delayed-release dosage forms that significantly delay the release of the drug after its administration; (2) sustained-release dosage forms that prolong drug level in biological fluids and tissues; and (3) controlled-release dosage forms that release the drug at a constant rate.
Examples for release of the drug at a constant rate are the diffusion method Rubinstein, A. and Robinson J. R. (1986) in: Progress in Clinical Biochemistry and Medicine, Springer-Verlay!, the osmotic pressure method Theeuwes, F. (1975) J. Pharm. Sci. 64: 1987-1990! and the ion-exchange method Schach, E. H. (1983) in: Controlled Drug Delivery, Vol. 1 (ed. Burck, S. D.) Florida CRC Press, pp. 149-161!.
Several approaches are sued in the design of sustained-release dosage forms, such as the mechanical approach and the prodrug approach. The mechanical approach is usually adopted when there is a demand for a large quantity of the drug over an extended period of time. Use of prodrug is most convenient when release of the drug at a specific target is desired. In this system, the drug in its active form is only obtained after chemical or enzymatic modification of the prodrug at the biological target site.
In order to deliver a drug to its target and to release it there at a constant rate, several methods have been developed, for example particulate methods such as microencapsulation, nanocapsules, drug embedded in matrix and microspheres Robinson, T. R. (1985) in: Recent Advances in Topical Drug Delivery: Rate Control, (ed. Nimmo, W. S.) Edinburg, Churchill Livingstone, 71; Friedman, M. (1973) in: Sustained Release Granules, Thesis, The Hebrew University of Jerusalem!. Other methods involve the use of liposomes (such as for SOD and catalase Tyrrell, D. A. et al. (1976) Biochim. Biophys. Acta 57: 359-367; Turrens, J. F. et al. (1984) J. Clin. Invest. 73: 87-95!) erythrocytes Inhaler, G. M. (1983) Pharmac. Ther. 20: 151-155!, monoclonal antibodies Yelton, D. E. and Scharff, M. D. (1981) Ann. Rev. Biochem. 50: 657-680! and magnetic fields as a targeting tool Hsieh, D. S. et al. (1981) Proc. Nat. Acad. Sci. USA 78: 91863-91868!.
Many attempts to interfere with the oxidative reactions by using novel antioxidants and by removing the reactive species have been carried out and are reviewed in the literature see, for example, Halliwell, B. et al. (1992) J. Lab. Clin. Med. 119: 598-620!. The limited success reported in many of these attempt resulted, in part, from the difficulties in introducing the antioxidants into cells and biological tissues and thus increases their level at the needed site. In order to prevent oxidative stress antioxidants have to be present in high concentrations and for long periods of time at their biological targets. The present invention is directed to such sustained release pharmaceutical dosage forms.